Evasion of programmed cell death (apoptosis) is one of the hallmarks of oncogenesis and many chemotherapeutic agents rely on the activation of apoptosis to induce cancer cell death. A critical step during the initiation of apoptosis is the induction of mitochondrial outer membrane permeability (MOMP) and the release of cytochrome c by the creation of pores in the mitochondria, orchestrated by the BCL-2 apoptosis-regulatory family. Recent data suggest that inner mitochondrial membrane-localized proteins (HAX1, OMI-HtrA2, and PARL;the HOP complex) possess antiapoptotic properties, and the inner mitochondrial membrane protein OPA1 has been shown to regulate cytochrome c release. Myeloid leukemia factor 1 (MLF1) was first identified as a fusion partner with nucleophosmin (NPM), expressed as the chimeric protein NPM-MLF1, in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The main hypothesis of this proposal is that MLF1 physically/functionally interfaces with both the intrinsic apoptotic cascade and the HOP mitochondrial protein complex to facilitate cytochrome c release to induce apoptotic cell death. This hypothesis will be addressed in two Specific Aims: 1) To elucidate the mechanisms by which MLF1 interacts with the classical intrinsic and the novel HAX1/OMI- HtrA2/PARL (HOP) apoptosls-control pathways to regulate mitochondrial cytochrome c release and induce apoptotic death. 2) To generate and characterize an in vivo NPM-MLF1-mediated leukemia model to further examine the mechanisms of MDS/AML pathogenesis, and as a tool to test anti-leukemia therapeutic approaches. To define those motifs of the MLF1 protein essential for interaction with BCL-2 family proteins and the HOP complex during the regulation of apoptosis, a series of MLF1 mutants will be employed. Mice, cells and mitochondria lacking MIf1, or other selected apoptosis regulators, will be used during these studies to help ensure the physiological relevance of our findings. This proposal seeks to also determine if the protein-protein interactions between MLF1 and BCL-2 family members or HOP proteins can be exploited for anticancer therapeutics. Finally, we propose to investigate the role of NPM-MLF1 in leukemogenesis by creating mice engineered to express an Npm-MLF1 fusion protein, employing a conditional inverter knock-in methodology. PUBLIC HEALTH RELEVANCE: In addition to furthering our basic understanding of apoptosis-regulatory pathways, elucidation of the role of MLF1 in cell death control may identify the protein as a target for anticancer drug design. Generation of an Npm-MLF1 mouse model of MDS/AML pathogenesis would serve as an experimental tool to assess the effectiveness of both existing as well as novel antileukemia therapies.